Isolation and characterization of SAP and CRP, two pentraxins from Pangasianodon ( Pangasius) hypophthalmus

• Published in: Fish & shellfish immunology 2010-05, Vol.28 (5), p.743-753
• Main Authors: Huong Giang, Duong Thi, Van Driessche, Edilbert, Vandenberghe, Isabel, Devreese, Bart, Beeckmans, Sonia
• Format: Article
• Language: English
• Subjects: Aeromonas hydrophila; Aeromonas hydrophila agglutination; Affinity chromatography purification; Agglutination; Amino Acid Sequence; Animals; Bacteria - metabolism; Blotting, Western; C-reactive protein (CRP); C-Reactive Protein - chemistry; C-Reactive Protein - genetics; C-Reactive Protein - isolation & purification; C-Reactive Protein - metabolism; Catfishes - genetics; Catfishes - metabolism; Chromatography, Gel; Circular dichroism (CD); Cross Reactions; Edwardsiella ictaluri; Edwardsiella ictaluri agglutination; Electrophoresis, Polyacrylamide Gel; Erythrocytes - metabolism; Escherichia coli; Haemagglutination; Hypophthalmus; Hypothalamus - metabolism; Micrococcus lysodeikticus; Molecular Sequence Data; Pangasianodon; Pangasianodon ( Pangasius) hypophthalmus; Pangasianodon hypophthalmus; Pangasius hypophthalmus; Pentraxin; Rabbits; Sequence Alignment; Serum amyloid P component (SAP); Serum Amyloid P-Component - chemistry; Serum Amyloid P-Component - genetics; Serum Amyloid P-Component - isolation & purification; Serum Amyloid P-Component - metabolism; Spectrum Analysis
• ISSN: 1050-4648; 1095-9947
• DOI: 10.1016/j.fsi.2010.01.007

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine–Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca 2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 μg SAP and 56 μg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1–2 mM), respectively. Circular dichroism studies revealed that antiparallel β-pleated sheets are dominating the secondary structure. Upon removing the Ca 2+ ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.